Method of hydroxylating steroids in the 21-position



Unite tates METHGD F HYDRGXYLA'ITNG STEROIDS IN THE ZE-POSITION No Drawing. Application July 6, 1956, Serial No. 596,164

Claims priority, application France .iuiy 21, 1955 8 Claims. (Cl. 195-51) This invention relates to a method of introducing by the action of specific microorganisms, a hydroxyl group at the 21-position of steroids of the pregnane series.

Most of the hormones of this series, such as desoxycorticosterone, cortisone, hydrocortisone, A -dehydrocortisone or prednisone, A -dehydrocortisol or prednisolone, have a hydroxyl group at the 21-position.

One member, the 21 -hydroxy pregnane, 3,20-dione can be used as starting material for derivatives that have a certain anaesthetic effect.

The hydroxyl at the 21-position is ordinarily introduced by chemical means, but the operation is not always successful, particularly if the pregnane is without a hydroxyl at the 17-position, because, in this case, the bromine, which is to be introduced at the 21-position to be-later replaced by a hydroxyl, becomes also fixed at the l7-position.

It is one of the objects of the present invention to introduce a hydroxyl group at the 21-position of various saturated and unsaturated steroid compounds and substituted steroid compounds of the pregnane series by eans of a new, single step process.

It is another object of this invention to provide substituted and unsubstituted 21-hydroxy pregnanes from new starting materials or from starting materials which hitherto have not been used in the preparation of these compounds.

It is a further object of the invention to introduce a hydroxyl group at the 2l-position of various steroid compounds of the pregnane series by subjecting such compounds to the action of a specific, herein claimed fungus, or to the action of oxidizing enzymes isolated from cultures of this fungus.

It is a still further object of the invention to provide a fungus strain that is particularly active in the formation of 2l-hydroxy pregnanes.

These and other objects and advantages of the present invention will appear more clearly from the herein following detailed description and from the appended claims.

The use of other, different microorganisms for the biological hydroxylation at the 2l-position has been previously described (Meystre et al., Helv. 1954, 37, 1540).

We have now found that 2l-hydroxy pregnanes are obtained at particularly high efficiency by subjecting various steroid compounds of the pregnane series to the action of a fungus species, Colletotrichum Lindemurhianum, belonging to the Melanconiaceae family of the genus melanconiales, or by exposing these compounds to the action of oxidizing enzymes isolated from cultures of these microorganisms. It should be noted that all species of the melanconiaceae family are not equally efieetive in introducing the hydroxyl group at position 21. Difiereut species of Collerotrichum Lindemuthianum have been isolated and carefully distinguished.

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Generally, descriptions of several of these species can be found in papers on mycology and in scientific literature.

However, we have been able to isolate, from a bean pod attacked by anthracnosis, a particularly active strain, as will be demonstrated further below. Cultures of this strain are kept in the laboratories of the assignee of this application and have been designated as strain U 230.

PROPERTIES OF STRAIN U 230 On an appropriate agar medium based, say, on a decoction of potatoes and saccharose, strain U 230 shows, after 6 to 10 days of growth at a temperature of approximately 24 C., a mycelium of dark color, the conidiophores of which carry small black conidia of oblong, more or less inwardly curved shape without secretion of exocellular pigment difiusing into the agar.

The herein claimed process comprises growing a first culture of 'Colletotrichum Lindemuthianum on a suitable agar medium, for example, an agar medium including saccharose and a decoction of potatoes, collecting the conidia of this first culture and inoculating therewith in sterile manner a suitable culture medium contained, for instance, in Erlenmeyer flasks. This second fermentation is carried out at a suitable temperature with the flasks mounted on a shaker. After a period varying between 1 and 10 days, a suitable solvent solution of the pregnane that is to be hydroxylated is introduced into the flasks, which are then incubated for a period ranging from 12 to 50 hours. Prior to recovery, the effectiveness of the hydroxylation is determined in an aliquot part of the culture medium. The isolation is carried out by means of one of the conventional extraction methods, followed by chromatographic purifi cation, recrystallizations or conversion into specific derivatives, permitting the separation of the desired compound from secondary fermentation products or from unconverted starting material. In certain cases and depending upon the nature of the steroid, the herein claimed microorganism may attach one or several additional hydroxyls at other than the 2l-position in a certain amount of the steroid.

The fluid culture medium for Colie'totrichum Lindemuthianum comprises preferably a source of carbohydrate, such as glucose, saccharose or starch. In addition, a source of mineral nitrogen, such as nitrates, ammonium salts, etc., may be used. Of course, mineral nitrogen may be complemented or substituted by organic nitrogen sources, such as protein hydrolysate, soybean flour, cottonseed flour, etc. Mineral salts, such as phosphate, potassium salts, iron salts, magnesium Salts or calcium salts may be used. A buffer, such as calcium carbonate, may be required at times, although, generally, the pH value does not change very much during fermentation.

During the process which, on a commercial scale is preferably carried out in tanks equipped for submerged aerobic fermentation, the medium is agitated and aerated in the conventional manner used in the production of antibiotics. Sometimes, an antifoaming agent is required.

In order to carry out the process of this invention, the steroid compound which is to be hydroxylated at the 21- position can be introduced into the fermentation medium in form of finely divided solids or dissolved in a solvent which does not interfere with the growth of Colletotrichum Limlemzzthianum. The steroid compound may be added at the start or during the course of the fermentation. Moreover, hydroxylation may also be carried out by subjecting the pregnane in question-to the action'of the fermented juice or to the action of an enzyme exthis method can be used to control the conversion during a the operation. Isolation is then carried out by conventional methods. v p v The following examples are presented to illustrate the invention without intent, however, to thereby limit the scope of the appended claims,

Example .7. Preparation of N-dehydrbcortisone (170:, 21 -dihydrxy A -pregnadiene 3,11,20-tri0nc) (i1) PREPARATION OFI'FMHYDROXY ALLPREGNADIENE 3,11,20-TRIONE This compound is prepared from 17a-hydroxy 2,4-di bromo pregnlane 3,11,20-trione, as described in Example 4 of applicants copending U. S. patent application Serial No. 360,878 of June 11, 1953.

5 gr. of the dibrominated derivative, M. P.=269270 C., [ct] =-|-62 (c.=0.5%, acetone) 'are introduced into 20 cc. of redistilled collidine. A stream of nitrogen is passed through the solution, and about 15 cc. of collidine are distilled 01f within 15 minutes While stirring. After cooling, the residue is taken up with 50 cc. of a mixture of ice-Water and 15 cc. concentrated hydrochloric acid; The resulting precipitate is separated and washed in Water until the wash Water no longer gives a chloride reaction. It is then desiccated and dried at 40 C.

U. V. spectro-graphy at 230 m indicates that the product comprises 50% of17a-hydroxy A -pregn adiene 3,11, 20-trione whichis isolated by chromatography on alumina. Thisproduces about 1.7 gr. of the desired diene, M. P.==244 C., -[a] =+142 (c.=1%, chloroform). For purification, the diene is pasted with 3 volumes of absolute ethanol while stirring and refluxing. Cooling and recovery result in'about 1.2 gr. of the pure product,

M. P.' '245 C, (the product melts, recrystallizes and melts again at about 280 C.) [a] =+149 (c.=1%,' chloroform).

Microanalysis: C21H2e04=342.'43.

Calculated: C% 73.66% C; 7.65% H; 18.69% 0 Found: 73.6% C; 7.7% H; 18.7% 0.

This new product is soluble in dimethylformamide, dioxane, sparsely soluble (from 1% to 2% at ordinary temperature) in methanol, methylcellosolve; methyl ethylketone, insoluble in water and isopropyl ether.

(1)) BIOLOGICAL HYDRO'XYLATION Colletotrichum .Linde'm'azhianum is cultivated for days at 24 C. on an agar medium c'ompn'sing 2% of saccharose and 20% of a potato decoction; 7 The conidia are collected in distilledwater. The suspension thus obtained is used for sterile inoculation of a 1 liter Erlenmeyer flask containing 100 cc. of the following substrate:

' Gr Pure glucose 10 Malt extract V 5 Soybean flour; 10 Sodium chloride 5 Dry corn steeping 1iquor 5 Calcium carbonate 1 Tap water to 1000 cc.

' Prior to inoculation, the pH is adjusted to 6.8-7.0 by meansof potassium hydroxide and the substrate is sterilized-by heating to 120 C. for thirty minutes. After 5 days of incubation at 240 C. while shaking (85 oscillations per minute, 8 cm. stroke), 1 cc. of a 1% acetone solutionxof 17a-hydroxy A -pre'gna'diene 3,11,20-trione, prepared according 'to the foregoing description is added tothe 100- cc. culture- A 24 hour continued incubation under the same conditions produces 3,11,20-triketo l7a,21-dihydroxy A -pregnadiene at a yield of 50%, as found by means of the following paper-chromatographic determination: 50 cc. of culture broth are filtered and the mycelium is washed twice with 5 cc. of acetone which is added to the filtrate. The mycelium is then extracted twice with 50 cc. ofchloroform, land the foregoing filtrate is extracted twice with these 100 cc. of

chloroform and then twice more, each time with 20 cc. of chloroform. The combined chloroform extracts are first washed with an aqueous bicarbonate solution, then with Water, and are dried over magnesium sulfate and vacuum evaporated-to dryness. The residue is taken up with 1 cc.

of methanoland used for paper chromatography. For this purpose, Whatman No. 1 paper is Washed in benzene .and methanol. Prior to chromatography, the paper is immersed in a 30% propylene-glycol solution. The solu- 7 tion having dripped oh, the chromatography is carried out by using for the steroid propylene-glycol saturated toluene with a development of from eight to fifteen hours. The spots are detected either by fluorescene in U. V. light or by color stain reaction.

By inserting the dried sheet' between a source of filtered ultraviolet (A max.=2400A.) and a fluorescent screen, the places at which there (are steroids absorbing the U. V. become visible on the screen as violet spots on a luminous background.

The color'stain reaction is that of.Mader and Buck,

Anal. Chem, 1952, 24, .666, with triphenyl tetrazolium chloride which gives a red coloring on white ground with steroids which have the keto function RCOCH2OH Control experiments with several successive dilutions of A -dehydrocortisone in the same solvent indicate that the starring product has been converted into A -dehydrocortison'e at a yield of about 50%.

Example 2.-Preparati0n of N-dehydr'ocorfisone Fermentation is carried out as described-in Example 1(b), but operating with 400mg. of 17-hydroxy A pregnadiene 3,11,20-trione and using an amount of culture medium that is 40 times that of Example 1. These 4 liters of culture medium produce, afterthe mycelium hasbeen filtered, washed in acetone andextracted with chloroform identical with the product described in the literature 7 (Herzog, et;al., Science,.1955,121, 176) Example 3.Mici0b i0l0gical hydrorylation of progesterone The method described in Example 3 is followed, but 3,20-pregn'anedione is used instead of progesterone. P5 per chromotagraphy indicates that compounds have formed" which have a hydroxyl group at the 21-position.

It will be obvious from the foregoing examples that it is. possible to alter the culture medium,conditions of incubation and methods of determination and extraction of gr. to 1.6 gr. of a material'o'f greasy 1 the desired product without exceeding the scope of the present invention.

Moreover, aside from the use of the basic U 230 strain of Colletotrichum Lindemuthianum, this invention relates to natural mutations thereof and mutations induced by X-rays, ultrasonic treatment, nitrogenated yperite.

A culture of U 230, i. e., the hydroxylating strain of the present invention has been deposited in the American Type Culture Collection, Washington, D. C., and added to its permanent collection of microorganisms. The said deposited culture has been assigned the catalogue or accession number 12611 by said American Type Culture Collection.

We claim:

1. The method of converting a steroid of the series consisting of saturated and unsaturated pregnanes and substituted pregnanes into a 21-hydroxy steroid, which comprises fermenting a culture of Colletotrichum Lindemuthianum under submerged-aerobic conditions, adding said steroid to the fermenting culture, continuing the fermentation for a period suflicient to convert at least part of said steroid into the respective 2l-hydroxy steroid, extracting the culture with a water-immiscible solvent, and separating 21-hydroxy steroid from the extract.

2. The method according to claim 1, whereby said 21- hydroxy steroid is separated chromatographically.

3. The method according to claim 1, whereby said 21- hydroxy steroid is separated by recrystallization.

4. The method according to claim 1, whereby said 21- hydroxy steroid is separated by conversion into a readily separable derivative.

5. The method according to claim 1, which comprises subjecting 17a-hydroxy A -pregnadiene 3,10,20-trione to the action of a U 230 Colletotrichum Lindemuthianum culture fermenting under submerged-aerobic conditions in a fluid fermentation medium, separating the mycelium of said Colletotrichum Lindemuthianum from the fluid fermentation medium, extracting the mycelium and the fluid fermentation medium with chloroform, combining the extracts, evaporating to dryness, degreasing the residue thus obtained with isopropyl ether and recovering A -dehydrocortisone chromatographically from the residue.

6. The method according to claim 1, whereby said steroid to be converted into a 21-hydroxy steroid is progesterone.

7. The method according to claim 1, whereby said steroid to be converted into a 21-hydroxy steroid is pregnanedione.

8. The method of claim 1, wherein one of said steroids is subjected to the action of oxidizing enzymes isolated from a U 230 Colletotrichum Lindemuthianum culture.

Vischer et al.: Experientia IX, 10, 1953, pages 371-372.

Meystre et al.: Helvetica Chimica Acta, 37, 1954, pages 1548-15 53.

Zafiaroni et al.: Experientia 11, 1955, page 219. 

1. THE METHOD OF CONVERTING A STEROID OF THE SERIES CONSISTING OF SATURATED AND UNSATURATED PREGNANES AND SUBSTITUTED PREGNANED INTO A 21-HYDROXY STEROID, WHICH COMPRISES FERMENTING A CULTURE OF COLLETOTRICHUM LINDEMUTHIANUM UNDER SUBMERGED-AEROBIC CONDITIONS, ADDING SAID STEROID TO THE FERMENTING CULTURE, CONTAINING THE FERMENTATION FOR A PERIOD SUFFICIENT TO CONVERT AT LEAST PART OF SAID STEROID INTO THE RESPECTIVE 21-HYDROXY STEROID, EXTRACTING THE CULTURE WITH A WATER-IMMISCIBLE SOLVENT, AND SEPARATING 21-HYDROXY STEROID FROM THE EXTRACT. 